1. Develop and evaluate potential alternatives to antimicrobials and other intervention products and strategies to control and reduce foodborne pathogens in poultry and swine. 1a. Select, chemically synthesize and screen antimicrobial peptides (AMP) for ability to kill Campylobacter spp. in vitro. 1b. Evaluate in vitro and in vivo efficacy of the most active AMP products expressed in yeast or plant vectors and, as necessary, develop encapsulation procedures for enhanced stability, site-directed delivery, efficacy and storage of the protein. 1c. Conduct Campylobacter challenge trials in broiler chickens to determine the ability of oral administration of AMP products to reduce Campylobacter colonization and utilize 16S rRNA sequencing to determine the effect of the peptide products on the overall cecal microbiota. 2. Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. 2a. Identify epitopes of the pathogens from peptide microarrays using chicken serum samples from the field. 2b. Construct and express of the epitope containing genes in an Escherichia (E.) coli expression system and purify of the recombinant proteins. 2c. Assay of the immune response in broilers to these recombinant proteins. 2d. Examine populations of bacteria in chicken gastrointestinal tract (GIT) after vaccination to determine the effects of vaccines on the microbiota of the GIT. 3. Employ molecular methods to scientifically guide the development of intervention and mitigation strategies for Campylobacter spp. during poultry production and processing. 3a. Continue Whole Genome Sequence (WGS) comparisons of 50 genetically distinct C. jejuni isolates that vary in their ability to colonize broiler chickens. 3b. Evaluate and refine an atmospheric cold plasma based antimicrobial packaging system (ACP) using genetically diverse C. jejuni isolates, and subsequently perform proteomic analyses to determine the mechanisms associated with die-off. 4. Survey local, pastured-raised, multi-commodity, antimicrobial growth promoter (AGP)-free poultry farms for the presence and variability of common foodborne pathogens as well as to establish changes in the microbial ecology along the “local” farm to fork continuum. 4a. Employ microbiological methods for the recovery of Campylobacter spp. from broiler feces during production, environmental samples, and processing samples. 4b. Perform microbiome analyses on samples to establish changes in the microbial ecology along the “local” farm to fork continuum. 5. Develop, refine, and implement improved methods for the detection and recovery of Campylobacter spp. and other potentially emerging foodborne pathogens, so that the methods meet the needs of associated regulatory agencies. 5.a. Continue collaboration with Pathsensors Inc. for the development of rapid and specific antibody biosensor..." 5.b. Compare traditional selective media/recovery technologies and a non-selective/filtration (Campycheck) methodology..." Please see objective 3 of project plan 070-00D for complete subobjectives for what is now objective 5 of this project.
Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) Develop and evaluate antimicrobial peptides (AMP) as potential alternatives to current antibiotics to control and reduce foodborne pathogens in poultry, and 2) Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. Specifically, we will select, chemically synthesize, and screen a panel of natural and synthetic AMP for ability to kill Campylobacter spp. in vitro. The genes for expression of the most effective AMP will be coupled to the genes encoding a well-defined bacteriophage receptor binding protein (RBP) to enhance specificity of the AMP for Campylobacters and the AMP-RBP construct will be expressed in a yeast for enhanced production of the protein for evaluation of efficacy. Encapsulation protocols will also be developed for enhanced stability, storage and site-directed delivery of the expressed AMP-RBP product and subsequent Campylobacter colonization challenge trials will be conducted in chicken and swine to evaluate the efficacy of the treatment and determine its overall impact on the gastrointestinal tract (GIT) microbiota. In our second approach (vaccine development) we will identify specific epitopes of the pathogens from peptide microarrays using serum samples from mature commercial chickens. We will then construct and express the epitope containing genes in an Escherichia coli expression system, purify the recombinant proteins, and assay the immune response in broilers to the recombinant proteins. Finally, we will examine the populations of bacteria in the chicken GIT after vaccination to determine the effects of the vaccines on the microbiota. Several knowledge gaps exist regarding the persistence and transmission of Campylobacter during the poultry production/processing continuum. To address these gaps, the proposed research will 1) employ molecular methods to characterize and scientifically guide the development of intervention/mitigation strategies for Campylobacter during poultry production/processing; 2) survey local, pastured-raised, antimicrobial growth promoter-free poultry farms for the presence and variability of common food pathogens; establish changes in the microbial ecology; and establish background levels of antimicrobial resistant pathogens in poultry production devoid of exogenous sources of antimicrobial drugs; and 3) continue to develop and test rapid, specific, and sensitive detection and cultural methods for Campylobacter to assist in meeting the needs of both regulatory agencies and the poultry industry.
ARS researchers in Athens, Georgia, continued to conduct research on development of selected antimicrobial peptides for reduction of Campylobacters and other pathogens associated with poultry. The minimum inhibitory concentrations of the peptides required to kill pathogens have been determined and toxicity testing for the peptides has been completed. Recent work has focused on determining stability of the peptides to protease and other enzymes. Work is continuing on production and purification of recombinant Salmonella proteins for vaccination of broiler chickens against Salmonella. More than 10 purified recombinant proteins have been obtained. Other research has been conducted to investigate the microbiome of poultry meats after cold plasma and antioxidant treatments. The EcoPlates™ were used to assess meat microbiome in response to an individual substrate as carbon sources. Results show significant differences in the bacterial communities of the poultry meats after treatment in the study. Also, results show bacteria in the meat communities could not metabolize the following substrates for their growth: phenylethylamine, lactose, glycerol phosphate, 2-hydroxybenzoic acid, hydroxybutyric acid, ketobutyric acid or malic acid.
1. Bacterial community in poultry meat samples. ARS researchers in Athens, Georgia, completed studies that examined the microbial community in poultry meat samples. EcoPlates™ from Biolog, Inc. were used to characterize bacterial communities at the physiological level. Findings indicated that the bacterial communities in the samples after cold plasma and anti-oxidant treatment metabolized test substrates at different rates thereby showing that the poultry meat samples contained different amounts and types of bacteria. Seven of these test substrates could not be metabolized by the bacteria in the samples. Future research will determine if any of seven non-metabolized substrates can be potential new candidates for preservatives of fresh poultry meat.
Yeh, H., Line, J.E., Hinton Jr, A., Gao, Y., Zhuang, H. 2019. The effect of rosemary extract and cold plasma treatments on bacterial community diversity in poultry ground meats. Heliyon. 5:e02719. https://doi.org/10.1016/j.heliyon.2019.e02719.
Yeh, H., Amad, A. 2020. Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). Current Microbiology. 77:1647–1652. https://doi.org/10.1007/s00284-020-01965-w.